The Bioprep5 robotics system

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The Bioprep5 robotics system The development of an automated grape analysis sample preparation unit Leigh Francis, Wies Cynkar, Mariola Kwiatkowski and Peter Høj

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HIS ARTICLE provides an overview of work that has

been conducted by The Australian Wine Research Institute (AWRI) as part of the research program of the Cooperative Research Centre for Viticulture (CRCV), to automate laboratory procedures for grape colour (total anthocyanins) and glycosyl-glucose measurement. The history of the relationship between the AWRI and the robotics company Advanced Rapid Robotics Manufacturing (ARRM) in this process is outlined. This project was supported by the Australian Government’s Cooperative Research Centre’s program and the Grape and Wine Research and Development Corporation (GWRDC). The close working association between the organisations culminated in the recent commercial release of the ARRM Bioprep5 robotics system. The Bioprep5 is a compact and fully automated unit that has been primarily designed to carry out grape sample preparation steps for the colour assay. It is, however, a flexible system that is not necessarily restricted to grape berry samples. It could thus be applied to other fruits or foods, as well as other matrices. A summary of investigations carried out recently by AWRI staff to assess the performance of the Bioprep5 robotics system is also provided in this article.

INTRODUCTION

By the early 1990s research efforts at the AWRI under the direction of Dr Pat Williams had determined that many grape-derived compounds giving rise to wine aromas are bound to sugars in a flavourless glycoside precursor form in the grape berry (see Williams et al. 1992). Having made this important observation, a relatively simple laboratory assay was developed to quantify the total amount of glycosides in grapes, juices or wines, and was named the G-G (GlycosylGlucose) assay. This assay was refined in the period from 1991 to 1994, with an initial report describing an enzymebased variant of the assay (Abbott et al. 1992), followed by a disclosure of a modified, well-validated assay applied to juice or wine glycosides (Williams et al. 1995), and finally a series of investigations were published in a paper detailing a method for application to grape berries, particularly for red Note that this article does not constitute an endorsement of the Bioprep5 robotics system. Prospective buyers should make an independent assessment of the Bioprep5 robotics unit, which is subject to regular improvements by ARRM.

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grape varieties (Iland et al. 1996). In the later report a description of assaying anthocyanin concentration using the same sample preparation protocol as used for the G-G assay was disclosed. Dr Patrick Iland, then at the University of Adelaide, was a leading figure in the development of these assays. During this period the assay was applied in Australia, for example in a national survey to assess its utility (Francis et al. 1998, a,b), and overseas, notably by Dr Bruce Zoeckein and colleagues (for example McMahon et al. 1999, Mansfield and Zoecklein 2003), who have developed some modifications of the G-G assay (Zoecklein et al. 2000, Whiton and Zoecklein 2002). Both the G-G assay and the colour assay for grapes require the same sample preparation: extraction of a subsample of a thoroughly blended, homogenised berry sample using an ethanol solvent, followed by centrifugation. This sample preparation procedure, while quite simple, is fairly tedious and relatively time consuming, and constitutes the largest proportion of work required for the colour assay. The G-G assay also requires subsequent additional solid phase extraction and acid digestion steps, and a final enzymatic spectrophotometric analysis. While quite simple to carry out, the G-G assay is certainly multi-step and relatively lengthy. AUTOMATION OF THE G - G / COLOUR ASSAYS : ‘ THE G - G ROBOT ’

After having developed the colour and G-G assays by 1994, and in considering the routine analysis of the colour and G-G assays for both AWRI application and potential wine industry use, it was considered that some means of automating the procedures would be highly desirable. Both assays, to be successfully applied to grape berry samples, require quite large numbers of samples to be processed. The use of automation was an obvious avenue to be explored to increase throughput. In considering commercially available ‘off the shelf’ automated options, it was found that a number of companies were able to supply equipment to semi-automatically carry out the solid phase extraction step of the G-G assay, and there were some systems to take care of the enzyme spectrophotometric assay. However, there was no possibility of automating the grape processing step using available equipment, which is

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system. The estimated project completion date was August 1996. Fairly early in the development and design phase, ARRM and AWRI recognised that producing the integrated spectrophotometric analysis section of the system (for operation 4 in the aforementioned list), would require significant time and resource investments that were not warranted, because of the availability of off-line alternatives. The contract was varied by mutual agreement to remove this requirement. The design and construction phases of the project proceeded smoothly and the production stage of the project was completed by October 1996, whereby the unit was physically constructed but a number of essential aspects of the system, such as the controlling software, were still being developed. Progress was then slowed, with some substantial successes offset by a number of setbacks and delays during the next 12 months. By 1997, the grape sample homogenising section of the system, a component of the future Bioprep5 system, was largely completed. It was tested by AWRI staff and found to work well. However, further delays were experienced with one of the principals of ARRM leaving the company in 1998, which was soon after the retirement of Dr Pat Williams from the Institute in November 1997. This precipitated a review of the project which was close to being discontinued. After taking into account the tangible intellectual property generated through the ARRM, AWRI and CRCV investments, and following assurances from ARRM that issues of delays and technical capacity would be resolved with the new technical team involved, it was agreed by the three parties that the project should continue. This decision was made partly as a result of the way the original contract was written, with the agreement specifying that no further payments were required to be made until a completed, working system was delivered by ARRM. In continuing to develop the system, the ARRM team achieved some notable successes, overcoming a number of serious technical issues with some innovative and elegant designs. However, given the relatively modest funds available, it was becoming more and more evident that the project was larger and more complex than anticipated, both at the start of the project and at the time of the change in leadership of the project. Consequently it was decided to have an independent review of the system. In 2000, following a search for suitably qualified independent engineers, a Western Australian company was selected to carry out the review. This organisation had extensive

Figure 1. The ARRM Bioprep5 unit.

arguably the most labour intensive part of the procedure. It came to our attention in 1994 that an Adelaide-based robotics company, ARRM, had recently started business specialising in laboratory automation. In discussing our needs with the ARRM team, including its CEO George Kraguljac, it was suggested that ARRM could develop and supply a system to automate the entire G-G process, from the grape sample preparation to the enzymatic assay, in a single integrated unit. Dr Pat Williams negotiated a legal contract with ARRM which was signed off in June 1995, following due diligence checks. In the contract, ARRM agreed to develop and manufacture an automated system for the G-G and grape colour assays (the ‘GG robot’, more properly named during development ARRM 203). The contract specified detailed performance criteria, where it was agreed that the completed system would carry out automated, unattended operations involving: 1. Grape sample processing (homogenise a grape sample, sub-sample, record the weight, add ethanol, mix, and centrifuge); 2. Solid phase extraction; 3. Heating for acid digestion; and 4. Final enzymatic spectrophotometric analysis. For each of the four sections of the system a number of liquid transfer/addition steps were involved. The agreement specified the minimum number of samples that should be processed in a defined period, as well as levels of accuracy and precision that were required to be achieved by the completed

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Berry colour - Bioprep5 processing (mg/g fresh weight)

3.0 y = 0.995x + 0.019 R2 = 0.980

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0.0 0.0

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Berry colour - manual processing (mg/g fresh weight)

Figure 2. Relationship between the colour values of red grape samples processed by the ARRM Bioprep5 unit and the same grape homogenates processed by the manual method (n=58).

robotics and automation experience, and prepared a carefully considered report. In the review it was stated that a significant amount of work was still needed to complete the entire G-G robot system, and that of the sections of the system produced to date, the grape sample processing section was worthy of production as a stand alone unit. In early 2002, it was agreed by ARRM, AWRI and CRCV that the G-G robot project should be discontinued and that any further development be focused on a functional grape sample-processing unit, i.e. the Bioprep5 system. In hindsight, it was clear that with the limited resources available, the G-G robot project was too ambitious, considering the number and complexity of the steps in the G-G assay. THE BIOPREP 5 ROBOTICS SYSTEM

As a result of the original G-G robot development project, there were a number of successful innovations that were produced by ARRM working with AWRI staff. With the recognition that the grape sample processing section of the G-G robot was of value as a stand-alone unit, a team of ARRM engineers developed a prototype of a relatively small system. The unit was designed to produce liquid extracts from grape samples, which could then be analysed off-line by a spectrophotometer for colour, i.e. anthocyanin content, or processed further manually for G-G and other analyses. AWRI staff assisted in the design and production process for this system, and carried out testing and evaluation of the first prototype unit built. It should be emphasised that although the AWRI no longer has a direct contractual relationship with ARRM, a royalty on sales of the system is payable to the AWRI under the conditions of the original G-G robot contract, as the unit is a result of development work carried out on the larger system. The future relationship between ARRM, AWRI and CRCV is at present being discussed. The unit, named the Bioprep5 robotics system, compris-

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es a 16-place sample carousel, capable of holding 200-300g of each berry sample; a homogeniser unit; a pipetting system for liquid transfers; a rotating mixer unit; a balance; and a centrifuge, and is controlled by proprietary software on a personal computer. The system has been designed to produce a clarified grape extract that can be then taken manually for a reading in a spectrophotometer, to allow an accurate measurement of the colour value of grapes. The semiautomated prototype unit was assessed during October/November 2002 at the AWRI, and the unit was released commercially in a fully automated version by ARRM in November 2002 (Figure 1). The commercial system has been tested by AWRI staff in a number of trials. The performance was initially evaluated by assessing the berry colour results obtained after processing by the Bioprep5, in comparison to the colour data of the same sample taken for processing using the standard, manual method. In the first series of tests a berry sample was homogenised by the Bioprep5, with a subsample of the homogenate taken by the Bioprep5 unit and processed automatically, and another subsample of the homogenate taken manually and processed manually. Note that this test does not check the efficiency of the homogeniser unit of the Bioprep5, but we were confident of the efficiency of this step of the assay from earlier testing of the prototype unit. Fifty-eight grape samples were tested over four runs, carried out on different days. The grapes used were from several varieties (predominantly Shiraz and Cabernet Sauvignon, but with Merlot, Grenache, Touriga, Barbera, Tarrango, Pinot Noir samples and a single Pinot Gris), and had been stored frozen. The grape samples were selected to have varied colour values, and some of the samples were chosen because they had previously been found to be difficult to homogenise manually. The results of this test showed that there was no statistically significant difference between the two methods, with both giving virtually identical results (Figure 2), as indicated by the high coefficient of determination (R2) of the linear regression and the fact that the slope of the regression line was close to 1.0. The average percentage difference in colour values between pairs of samples processed by the Bioprep and manually was 0.6%, an outstanding result. A further test was undertaken whereby 10 grape berry lots were individually carefully manually subsampled, so that two close-to-identical berry samples were obtained from each lot. One sample of each of the 10 lots was processed using the Bioprep5 system, while the other was processed manually. This test takes into account the efficiency of homogenisation of the system. Once again, there was no significant difference between the two methods (by paired t-test, data not shown). To test the reproducibility of the unit in carrying out subsampling and subsequent processing, the system was set to sub-sample 16 times from the same homogenised grape sample and take the samples through to the final stage of

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processing. The mean anthocyanin concentration value for the 16 samples was 1.15 mg/g, with a minimum of 1.10 and maximum of 1.18 mg/g, resulting in a percentage standard deviation of 1.9%, which is comparable to the values that are obtained using manual processing. The throughput of the unit was also assessed. The system can multitask, so that while a set of homogenised grape samples is being extracted and then centrifuged, a further lot of new grape samples can undergo homogenisation. By carrying out staggered processing in this way, it was found that 48 samples could be processed in six hours. Using the manual method, only 20 samples would be the normal maximum that could be processed in this period by an individual analyst. For laboratories carrying out near infrared (NIR) scanning, where homogenisation of grapes, but not extraction, is the main sample preparation required, the Bioprep5 system can process 16 samples in an hour for offline scanning, with none of the tedium normally associated with these pre-analysis steps. The unit has straightforward software control, where variations in processing can be set, including settings for different types of grape samples, dependent on how large the berries are. The machine has been found to be able to carry out effective washing of the homogeniser and the pippettor to avoid any carry-over between samples, and has been configured so that if by chance a drop of grape material might fall to the balance during the weighing process, this weight is not recorded. Overall, the accuracy and precision of the Bioprep5 was found to be similar to that obtained with the manual method by a skilled analyst, with the advantage of increased rate of processing of a given number of samples. As the unit can run completely unattended, an analyst can carry out other tasks related to the samples during the processing time, such as NIR scanning or laboratory analyses (e.g. pH, TSS readings) on previously processed samples. The system was found to be simple to use and to load, and required minimal cleaning. Loading the system involved placing sample jars containing grapes onto the carousel, empty centrifuge tubes in the appropriate holder, and ensuring sufficient 50% aqueous ethanol and water was added to two reservoirs. Ergonomics were good, with easy access to the unit, and safety issues have also been carefully considered with protective clear plastic panels encasing the unit, with cut-off switches which are activated if a panel is accessed during a run. To date the AWRI has carried out successful processing on more than 156 samples using the system. For those wineries considering carrying out colour assays during or after vintage, or indeed other assays requiring berry homogenisation, the system provides a desirable and realistic alternative to manual processing of grape samples, with its attendant difficulties and reliance on relatively skilled analysts to avoid errors and inaccuracies. For those wineries currently using NIR, the Bioprep5 system is well worth considering, for scans requiring homogenised fruit.

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CONCLUSION

The collaboration of ARRM with the AWRI and CRCV to produce an automated system to process grape samples for analysis, has resulted in the successful development of a commercial product that performs to specifications. Dr Pat Williams deserves recognition for his conviction and strength of purpose in starting the project and in overseeing it through the earlier stages of its life. The outcome from many years of effort by the personnel involved in the project, in striving for practical solutions for the automation of a complex laboratory assay, has been a compact, robust machine that can reliably and reproducibly produce grape extracts for the analysis of important quality measures. The continued uptake by the wine industry of the grape colour assay, and indeed any future measurements requiring sample homogenisation, suggests that the Bioprep5 unit has a role in a commercial production environment, assisting wine companies to continue to improve wine quality. ACKNOWLEDGMENTS

Mark Gishen, George Kraguljac, Professor Sakkie Pretorius and Dr Jim Hardie are thanked for critical comments on this article, and the contribution of the GWRDC, Dr Jim Hardie, the CRCV and the AWRI Council, in particular Geoff Linton, during the course of this project is gratefully acknowledged. Dr Pat Williams deserves particular thanks for both his comments on this article and his leadership of the project until his retirement. REFERENCES

Abbott, N.A., Williams, P.J. and Coombe, B.G. (1992) Measure of potential wine quality by analysis of grape glycosides. Stockley, C.S., Johnstone, R.S., Leske, P.A. and Lee, T.H. eds. Proceedings of the eighth Australian Wine Industry Technical Conference; 25–29 October 1992; Melbourne, Victoria. Adelaide, SA: Winetitles; 1993: 72–75. Iland, P.G., Cynkar, W., Francis, I.L., Williams, P.J. and Coombe, B.G. (1996) Optimisation of methods for the determination of total and red-free glycosyl glucose in black grape berries of Vitis vinifera. Aust. J. Grape Wine Res. 2: 171-178. Francis, L., Armstrong, H., Cynkar, W., Kwiatkowski, M., Iland, P. and Williams, P. (1998) A national vineyard fruit composition survey—the G–G assay. Aust. Grapegrower and Winemaker (414a): 51–53, 55–58. Francis, I.L., Armstrong, H., Cynkar, W., Kwiatkowski, M., Iland, P. and Williams, P.J. (1998) The 1997 CRCV national vineyard fruit composition survey—Shiraz data. Australian and NZ Wine Industry Journal. 13: 377–379. McMahon, H.M., Zoecklein, B.W. and Jasinski, Y.W. (1999) The effects of prefermentation maceration temperature and percent alcohol (v/v) at press on the concentration of Cabernet Sauvignon grape glycosides and glycoside fractions, American Journal of Enology and Viticulture 50 (4) : 385-390. Mansfield, A.K. and Zoecklein, B.W. (2003) Effect of fermentation, postfermentation, and postbottling heat treatment on Cabernet Sauvignon glycoconjugates. American Journal of Enology and Viticulture 54 2 : 99-104. Williams, P.J., Cynkar, W., Francis, I.L., Gray, J.D., Iland, P.G. and Coombe, B.G. (1995) Quantification of glycosides in grapes, juices and wines through a determination of glycosyl glucose. J. Agric. Food Chem. 43: 121–128. Williams, P.J., Sefton, M.A. and Francis, I.L. (1992) Glycosidic precursors of varietal grape and wine flavor. Teranishi, R., Takeoka, G. and Güntert, M., eds. Flavor Precursors: Thermal and Enzymatic Conversions of Precursors ACS Symposium Series No 490. American Chemical Society: Washington D.C.; 74-86. Whiton, R.S. and Zoecklein, B.W. (2002) Evaluation of glycosyl-glucose analytical methods for various glycosides. American Journal of Enology and Viticulture 53 4 : 315-317. Zoecklein, B.W., Douglas, L.S. and Jasinski, Y.W. (2000) Evaluation of the phenol-free glycosyl-glucose determination, American Journal of Enology and Viticulture 51 4 : 420-423.

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